Summary Title: Vanadium in lung mitochondria oxidative stress Occupational exposure to vanadium (V) causes fibrotic lung injury, and circumstantial evidence suggests that V from non-occupational exposures could also contribute to lung fibrosis. Experimental studies show that V accumulates in mitochondria and causes oxidative stress, but the biology of V is poorly understood, especially concerning dose-response effects on accumulation in lung and lung mitochondria and associated toxicologic responses. Our preliminary data from patients with idiopathic pulmonary fibrosis (IPF) show that V is inversely correlated with selenium (Se) in IPF lungs and lung mitochondria. Se protects against occupational levels of V toxicity in rats, raising the possibility that if environmental V causes or potentiates fibrotic lung disease, then it may be possible to develop strategies to decrease environmental V exposure or decrease risk by nutritional intervention with Se. Thus, we propose a critical set of cell and animal experiments to define the dose- response relationship of V with lung mitochondrial V, Se, oxidative stress, respiratory functions and profibrotic signaling to test the plausibility that environmental V contributes to risk of lung fibrosis. Our first specific aim is to determine the dose-response effects of V on Se-dependent antioxidant systems, metabolism and redox signaling, in lung fibroblasts, a cell type relevant to lung fibrosis. Experiments will use ICP-MS to quantify cellular loading of V and effects on Se contents. Seahorse technology will determine respiratory responses to V. Fluorescent redox probes will measure mitochondrial H2O2 and superoxide production. An integrated analysis of redox targets, metabolomics and transcriptomics will test for V disruption of energy metabolism and mitochondrial signaling. Measures of cell proliferation, senescence and cell death will establish dose-response characteristics of fibroblasts to V concentration range from very low, non-toxic to toxic exposure levels. Our second specific aim is to determine the dose-response relationship of V with lung and lung mitochondrial responses. Experiments will show whether in vivo administration of V to mice decreases lung and lung mitochondrial content of Se and related mitochondrial activities. Experiments with two models of administration will be performed, i.e., an acute intranasal vanadium pentoxide exposure at doses previously shown to cause subsequent lung fibrosis and 4- and 20-month dose-response with V in drinking water to mimic environmental exposure. Mouse lung tissue and isolated lung mitochondria will be analyzed for V and Se contents to compare these with existing human lung and lung mitochondrial data. Functional assays will be performed including mitochondrial Se-dependent enzymes, markers of profibrotic signaling and lung histopathology. The results will have sustained impact by providing fundamental new information to link V exposure dose to lung and lung mitochondrial V content and provide key information concerning potential mechanisms of V effects on lung fibrosis.